JOURNAL OF MEDICAL MICROBIOLOGY

Volume 56, Issue 8

Research Article
 
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Chlamydophila psittaci genotype E/B

transmission from African grey parrots to
humans 
 Taher Harkinezhad1, Kristel Verminnen1, Caroline Van
Droogenbroeck1, Daisy Vanrompay1
  View Affiliations
 First Published: 01 August 2007 https://doi.org/10.1099/jmm.0.47157-0

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ABSTRACT
 
Thirty-six birds from a parrot relief and breeding centre, as well as the manager, were

examined for the presence of Chlamydophila psittaci. In the relief unit, 5 of 20 African

grey parrots showed depression, ruffled feathers, loss of weight and mild dyspnoea. The

birds received no antibiotic treatment. Birds of the breeding unit, 14 blue and gold

macaws and 2 green-winged macaws, were healthy. They received doxycycline at the
 start of each breeding season. The manager complained of shortness of breath but took

no medication. Using a nested PCR enzyme immunoassay (EIA), Cp. psittaci was

detected in the faeces of all five sick birds, as well as in a nasal and pharyngeal swab

from the manager. The veterinarian and her assistant became infected while sampling

the parrots, as pharyngeal and nasal swabs from both were positive by nested PCR/EIA

after visiting the parrot relief and breeding centre, but they showed no clinical signs of

infection. Bacteria could be isolated from three of five nested PCR/EIA-positive birds,

the manager and the veterinarian, but not from the veterinary assistant. Using

an ompA genotype-specific real-time PCR, Cp. psittaci genotype E/B was identified as

the transmitted strain. All breeding birds tested negative for Cp. psittaci. This is

believed to be the first report on Cp. psittaci genotype E/B transmission from parrots to

humans. In contradiction to genotype A strains, which are thought to be highly virulent

to both birds and men, the currently described genotype E/B strain apparently caused no

severe clinical symptoms in either parrots or humans.

Keywords

 EIA, enzyme immunoassay, 

 MOMP, major outer membrane protein

INTRODUCTION
 
Chlamydophila psittaci, an obligate intracellular bacterium, has six known avian

serovars, all considered transmissible to humans (Andersen, 1991, 1997; Vanrompay et

al., 1993). The serovars are identified by mAbs recognizing serovar-specific epitopes on

the major outer membrane protein (MOMP). These serovars correspond to six

genotypes that are readily distinguished using outer membrane protein A (ompA) gene
sequencing, RFLP analysis of the ompA gene (Vanrompay et al., 1997), or a recently

developed genotype-specific real-time PCR (Geens et al., 2005a).

Genotype A is endemic among cockatoos, parrots, parakeets and lories (Psittaciformes),

and is well known as a zoonotic agent. Genotype B is endemic among pigeons.

Genotypes C and D are mostly detected in non-Psittaciformes. They are highly

pathogenic to domestic poultry, and are acknowledged occupational hazards for

slaughterhouse workers and generally for all people in contact with poultry, especially

turkeys and ducks. Genotype E isolates were first isolated during an outbreak of

pneumonitis in humans in the late 1920s and early 1930s. Subsequently, genotype E

isolates have been obtained from a variety of avian hosts worldwide, including turkeys,

ducks, pigeons, ostriches and rheas. Genotype F was first obtained from an American

parakeet (Andersen, 1997), and 8 years later also from a Belgian fattening turkey (Van

Loock et al., 2005a).

Recently, a new genotype E/B has been described (Geens et al., 2005b), which has been

isolated from Italian urban pigeons, German commercial fattening Pekin ducks and

Belgian fattening turkeys. Genotype E/B reacts with both the serovar E- and B-specific

mAbs, and generates the ompA RFLP pattern characteristic for genotype E. However, it

differs from genotype E and B by a unique combination of a guanosine residue at

positions 1006 and 1021, and a cytidine at position 1022, which results in an A instead

of S at position 341 in the variable segment 4 of the MOMP. Genotype E/B can only be

distinguished from genotypes E and B by ompA sequencing or by the recently

developed genotype-specific real-time PCR (Geens et al., 2005a). In the present study,

the genotype-specific real-time PCR was used to examine the transmission of a Cp.

psittaci genotype E/B strain from African grey parrots to humans.

 
METHODS
 
Background.A parrot relief and breeding centre near Antwerp (Belgium) was visited

in order to investigate the presence of Cp. psittaci in the birds and in the manager. The

parrot relief unit housed 20 African grey parrots (Psittacus erithacus), received from

people moving houses, or elderly people unable to nurse their birds any longer. The

parrots were kept in separate cages. Additionally, seven pairs of blue and gold macaws

(Ara ararauna) and one pair of green-winged macaws (Ara chloroptera) were kept for

breeding. Relief and breeding birds were housed in clean, heated and separate, but

adjacent, rooms. Every day, the 56-year-old manager was handling the parrots in the

relief unit for ~2 h, cleaning their cages, hand-feeding and even nuzzling the birds. The

man felt healthy, although he complained of continued shortness of breath. He took no

medication and was a non-smoker. The breeding birds were treated with doxycycline

(Soludox; Eurovet) in the drinking water during the 14 days before each reproduction

period, and they were healthy. However, 5 of 20 (25 %) birds in the relief unit showed

depression, ruffled feathers, loss of weight and mild dyspnoea. They received no

antibiotic treatment.

Samples.All birds, as well as the manager, were sampled using dacron-tipped

aluminium-shafted swabs (Fiers), filled with 2 ml Cp. psittaci transport medium

(Vanrompay et al., 1992) or DNA-stabilization buffer (Roche) for chlamydial isolation

or detection of the ompA gene, respectively. As bird catching was to be avoided due to

stress, fresh droppings were collected from each cage floor, sampling five different

spots per cage. The manager was sampled by taking both pharyngeal and nasal swabs.

The veterinarian and her assistant were identically examined ‘by chance’ 7 weeks later

at the start of a psittacosis occupational risk study, in which they voluntarily

participated. In these two persons, serology was additionally performed using an ELISA
with avian recombinant MOMP as a target antigen (Verminnen et al., 2006). Humans

were sampled with informed consent. All swabs were kept on ice during transport and

stored at –80 °C until use.

Cp. psittaci identification and molecular characterization.The presence of

the Cp. psittaci ompA gene was examined using a nested PCR/enzyme immunoassay

(EIA), as described by Van Loock et al. (2005b). The nested PCR/EIA allows ELISA-

based detection of the amplified gene, which becomes biotin- and fluorescein-labelled

during the amplification reaction. The presence of viable Cp. psittaci was examined by

isolation in buffalo green monkey (BGM) cells, which identifies the organisms by direct

immunofluorescence staining (IMAGEN; DakoCytomation), as previously described

(Vanrompay et al., 1992). Cp. psittaci was characterized directly from the clinical

specimens using a genotype-specific real-time PCR, as described by Geens et al.

(2005a). The latter is a TaqMan probe-based real-time PCR able to distinguish

the ompA gene of all six currently described avian Cp. psittaci genotypes A to F, plus

the recently described genotype E/B.

RESULTS AND DISCUSSION

 
Cp. psittaci genotype E/B transmission from parrots to humans

In the parrot relief unit examined in the present study, nested PCR/EIA revealed 5 of 20

(25 %) birds were positive for Cp. psittaci, and Cp. psittaci could be isolated from the

faeces of 3 of these. The chlamydial ompA gene was detected in nasal and pharyngeal

swabs from the manager. Nasal and pharyngeal swabs from the veterinarian and her

assistant, examined by chance 7 weeks after visiting the parrot and relief centre, also

contained Cp. psittaci DNA. At that time, the Cp. psittaci IgG (H+L) antibody titres in

the veterinarian and her assistant were 1/960 and 1/240, respectively.
As in birds, the infection of the manager and the veterinarian was confirmed by

isolating viable bacteria. Cp. psittaci isolates from the parrots, the manager and the

veterinarian were all characterized as ompA genotype E/B. However, at that time, we

could not isolate live chlamydial organisms from the veterinary assistant, nor could we

type the strain directly from the clinical specimens, as the genotype-specific real-time

PCR remained negative. Of the three persons involved, the veterinary assistant was the

one with the least close contact to the parrots, as she was only handing over the swabs to

the veterinarian. In contrast to the other two persons, she never went into the cages. This

could explain why she was negative for isolation and only positive by our nested

PCR/EIA, which is able to detect one organism, while our genotype-specific real-time

PCR has a detection limit of 10 chlamydial organisms.

During a subsequent medical consultation, the manager was advised not to take

antibiotics because he apparently showed no severe clinical symptoms. Until now, his

clinical condition has not changed. However, the manager decided to close the parrot

relief unit, not only due to our findings, but also for economic reasons, as heating costs

became too high. All birds of the relief unit were treated with doxycycline and

subsequently given to close associates of the manager who were

specialist Psittaciformes bird breeders. So far, there have been no reports on sickness or

abnormal mortality in these birds. However, as previously mentioned by Heddema et al.

(2006), the maintenance of accurate records of bird-related transactions for at least 1

year should be recommended, and every new bird brought into a colony should be

tested for Cp. psittaci. Tracking records should include the species of bird, bird

identification, source of bird and any identified illness. Upon closure of the relief unit,

the manager became negative for the nested PCR/EIA and for Cp. psittaci isolation.
As in the birds and the manager, the infection caused no severe clinical symptoms in the

veterinarian and her assistant. In fact, the latter two persons were unaware of being

infected. Therefore, they received no antibiotic treatment. Both persons became

negative for the nested PCR/EIA and for isolation 10 weeks after visiting the parrot

relief and breeding centre. However, at that time, the antibody titres in the veterinarian

and her assistant were still 1/960 and 1/120, respectively.

Psittaciformes: a higher risk to human health than other bird species?

Birds of the order Psittaciformes seem to be highly susceptible to Cp. psittaci infection,

as most veterinary case reports are about such infections in Cacatuidae (cockatoos)

and Psittacidae (parrots, parakeets, lories), dealing with severe clinical signs, such as

eye discharge or swelling, laboured breathing, diarrhoea, poor appetite, lethargy,

‘fluffed up’ appearance, weakness, and even mortality (Bracewell & Bevan,

1986; Chahota et al., 2006; Kaleta & Taday, 2003). Many of the birds become

chronically infected but show no clinical signs until stressed. These birds often shed Cp.

psittaci intermittently and serve as a source of infection for humans and other birds. In

particular, Amazon parrots (88 %), macaws (87 %), budgerigars (81 %), cockatoos (80 

%), conures and Senegal parrots (78 %), African grey parrots and eclectus (75 %), grey

cheek parrots (70 %), lovebirds (68 %) and cockatiels (65 %) are highly infected, as

demonstrated by serology (Fudge, 1997).

However, we do not really know if Psittaciformes are more susceptible to Cp.

psittaci than are other bird species. It might be that the disease is simply noticed and

therefore diagnosed more often, due to the severe clinical signs in these birds. For

instance, when comparing epidemiological data of chlamydiosis in Psittaciformes and

pigeons, the prevalence seems to be comparable, ranging from 16 to 81 % and 23 to 85 

%, respectively (Fudge, 1997; Trávnicek et al., 2002). However, chlamydiosis in

pigeons is less severe, and mortality is due more to secondary infections, such as

salmonellosis and trichomoniasis.

In fact, the overall clinical picture in a given species is the result of strain virulence and

host immunogenetics. We do not know much about bird immunogenetics, but we do

know that Cp. psittaci genotypes A, B and F have been isolated from Psittaciformes.

However, Psittaciformes are most frequently infected with genotype A strains, which

are highly virulent, intensively excreted, and often cause mortality (Andersen,

1991; Vanrompay et al., 1997). Pigeons on the other hand, mostly become infected by

genotype B and sometimes genotype E strains. However, the term genotype only refers

to the sequence of the ompA gene, which encodes for the Cp. psittaci immunodominant

MOMP. We do know that the MOMP is a porin and a possible bacterial adhesin, but the

direct contributions of this protein to the virulence of the bacterium are not clear. We do

not know why Psittaciformes are most frequently infected with genotype A strains,

which apparently cause severe disease and mortality in these birds, and why pigeons are

most frequently infected by other genotypes that are apparently less virulent. The

question could probably be solved if we knew more about host immunogenetics,

bacterium–host cell interactions and bacterial virulence factors such as the type three

secretion system, which was recently discovered in Cp. psittaci (Beeckman et al.,

2006).

Impact of clinically unnoticed Cp. psittaci transmission to humans

Given the fact that Psittaciformes are most frequently infected by genotype A strains,

and the proven zoonotic transmission of genotype A strains (Heddema et al., 2006), we

were surprised to find genotype E/B in both parrots and humans. So far, genotype E/B

has only been discovered in pigeons, ducks and turkeys, and all these birds were

clinically healthy except for the turkeys, which showed mild respiratory symptoms. In
this study, psittacosis infection was actually unnoticed in all three persons involved. It

may be that the E/B strain was indeed less virulent, although zoonotic transmission

occurred. On the other hand, frequent exposure to Cp. psittaci could also explain the

rather asymptomatic course of infection in these persons. However, we do not know

anything about natural protection following Cp. psittaci infection in humans.

For Chlamydia trachomatis infection in humans, protection against infection with the

same ompA genotype is thought to last ~6 months. In animals, it has been demonstrated

that fattening turkeys can become infected with two different genotypes during one

production period of 15 weeks (Van Loock et al., 2005a). Cp. psittaci infection can

cause severe disease in humans. However, these cases are probably only the tip of the

iceberg. What lies underneath are less severe, clinically unnoticed infections, which are

misdiagnosed due to symptoms similar to those of other respiratory pathogens, or even

asymptomatic infections. The impact of these types of Cp. psittaci infections on human

health is difficult to determine. We can try to extrapolate our knowledge on avian

infections to human psittacosis. As in birds, carrier status might occur, as well as

pathogenic interactions with other respiratory pathogens (Van Loock et al., 2006a, b).

Frequent use of tetracyclines in Psittaciformes

Notwithstanding possible air contact with infected birds in the relief unit, all breeding

birds were Cp. psittaci-negative in both nested PCR/EIA and bacteria isolation. Regular

doxycycline treatment might have prevented spread of the infection to the adjacent

breeding unit. With the risk of developing tetracycline-resistant strains, as described for

the Cp. psittaci-related species Chlamydia suis (Dugan et al., 2004, 2007), this is surely

not to be recommended as a preventive strategy. However, there is no avian Cp.

psittaci vaccine; therefore, owners of Psittaciformes frequently use tetracyclines for any

case of respiratory disease, or even prophylactically. One of the problems is that it is

possible to buy these drugs on the internet, even without a prescription, as this is not

needed in every country. However, most pet-bird owners are unaware of the dangerous

situation that they might create by using these drugs frequently. They are ignorant of

tetracylines being the drugs of choice when treating human psittacosis, and the fact that

the mortality rate prior to the advent of antimicrobial treatment was ~15–20 %.

Information campaigns on antibiotic use in pet birds with respect to zoonotic agents are

urgently required, and worldwide access to antibiotics has to be restricted to

veterinarians and medical doctors.

Conclusions

The number of human psittacosis cases is underestimated. For accurate diagnosis in

both birds and humans, we recommend nucleic-acid-amplification tests, which

specifically detect Cp. psittaci at high sensitivity and allow molecular characterization.

Parrots are often infected with genotype A strains, which are thought to be highly

virulent for both birds and humans. The current report is believed to be the first on Cp.

psittaci genotype E/B transmission from parrots to humans. In contrast to reports on

genotype A zoonotic transmission, the present genotype E/B strain caused no severe

clinical symptoms in either birds or humans.

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